植物激发子肽工程菌株的构建及其对棉花黄萎病的防治效果  

Construction of Plant Elicitor Peptide Engineering Strain and Its Control Effect on Cotton (Gossypium hirsutum) Verticillium Wilt

作　　者：尹铭绅 武国寒 雷绳尾 王羽 牛秋红[1] YIN Ming-Shen;WU Guo-Han;LEI Sheng-Wei;WANG Yu;NIU Qiu-Hong(College of Life Sciences and Agricultural Engineering,Nanyang Normal University,Nanyang 473061,China)

机构地区：[1]南阳师范学院生命科学与农业工程学院,南阳473061

出　　处：《农业生物技术学报》2024年第8期1890-1898,共9页Journal of Agricultural Biotechnology

基　　金：河南省杰出青年科学基金(222300420014);河南省农业生物质绿色转化技术高校科技创新团队(24IRTSTHN036);河南省能源微生物资源可持续利用创新型科技团队;南阳师范学院研究生创新基金(2021CX012)。

摘　　要：棉花黄萎病一直是限制我国棉花(Gossypium hirsutum)产业可持续发展的重要障碍。植物激发子肽(plant elicitor peptide, Pep)可增强植物对多种病原菌的免疫反应。本研究旨在构建棉花黄萎病植物激发子肽工程菌株,探究蛋白诱导肽在激活植物免疫后的抗菌作用,最终筛选出对棉花黄萎病具有良好防治效果的工程菌株。首先,对棉花黄萎病致病菌—大丽轮枝菌(Verticillium dahliae)的植物免疫诱导蛋白基因PEL1 (pectate lyase 1)与PevD1 (protein elicitor 1)进行克隆,并构建重组质粒p HT43-PEL1和p HT43-PevD1。经酶切与测序验证后,将质粒导入实验室前期筛选获得的拮抗棉花黄萎病菌的芽胞杆菌(Bacillus sp.) T6中,构建工程菌株。通过SDS-PAGE对PEL1和PevD1异源表达进行验证,结果显示,分别获得PEL1过表达的工程菌株V3-T和PevD1过表达的工程菌株P18-T。将工程菌株P18-T注射棉花叶片15 min,其活性氧含量较对照组显著增加(P<0.01)。在棉花盆栽试验中,工程菌株P18-T作用感病棉花植株30 d,使棉花黄萎病发病率和病情指数显著降低(P<0.05),防治效果达到78.85%(P<0.05)。本研究获得1株高效对抗棉花黄萎病的工程菌株P18-T,为拓宽棉花黄萎病综合防控技术手段提供了基础资料。Cotton verticillium wilt has always been an important obstacle to the sustainable development of China's cotton(Gossypium hirsutum)industry.It has been shown that plant elicitor peptide(Pep)can promote immune responses in multiple plant families against many kinds of pathogens.The purpose of this study was to construct an engineering strain of plant immune-inducing peptide for cotton verticillium wilt,and explore the antibacterial effect of protein-inducing peptide after activating plant immunity,then screen the engineering strain with good control effect on cotton verticillium wilt.Firstly,the plant immune-inducing protein genes PEL1(pectate lyase 1)and PevD1(protein elicitor 1)in Verticillium dahliae,a pathogenic bacterium of cotton wilt disease,were cloned,and the recombinant plasmids pHT43-PEL1 and pHT43-PevD1 were constructed.After being verified by enzyme digestion and sequencing,the plasmid was introduced into Bacillus sp.T6,which was screened in the early study,to construct engineering strain.The heterologous expression of the immune-inducing peptide was verified by SDS-PAGE,and the activity of the pot experiment was verified,so as to further confirm the engineering strain with efficient control of cotton verticillium wilt.The results of SDS-PAGE showed that the 2 inducible peptides were successfully overexpressed in T6 strain,and the engineering strain V3-T with PEL1 overexpression and the engineering strain P18-T with PevD1 overexpression were obtained respectively.The engineering strain P18-T was injected into the leaves of cotton plants for 15 min,and the active oxygen content increased significantly(P<0.01).In the cotton pot experiment,the engineering strain P18-T acted on the susceptible cotton plants for 30 d.Compared with the control wild strain T6 group,the incidence of cotton verticillium wilt in the P18-T treatment group decreased significantly(P<0.05),and the control effect reached to 78.85%(P<0.05).In this study,an engineering strain P18-T with high efficiency against cotton verticillium wilt

关 键 词：棉花黄萎病 植物激发子肽(Pep) 工程菌株构建 生物防治 

分 类 号：S432[农业科学—植物病理学]